Development and Validation of Voriconazole Concentration by LC-MS-MS: Applied in Clinical Implementation.

BACKGROUND: Voriconazole (VRZ) is a triazole antifungal used for treatment of invasive fungal infection, which is a life-threatening condition. Therapeutic drug monitoring is recommended for identifying the optimal dose in patients who have hepatic/renal impairment or reduced function of the CYP2C19 metabolizing enzyme. METHODS: One hundred microliters of sample plasma was extracted by protein precipitated with 200 µl of acetonitrile containing fluconazole as internal standard (IS). After vortexing and centrifugation, supernatant was dried and reconstituted with 100 µl of mobile phase (ACN: 0.1% formic acid in 10 mM Ammonium acetate) (50:50 v/v) before injected. The column was C18, 2.7 µm, 3.0 × 50 mm at flow rate of 0.5 ml/min with retention time of 0.5 and 0.75 min for VRZ and IS, respectively. The tandem mass spectrometer was set in multiple reactions monitoring (MRM) mode with the following transition; VRZ m/z 350.10→281.10 and 307.20→220.20 (IS). RESULTS: The accuracy and precision inter- and intra-day were less than 9%, over the range 0.05-10 µg/ml. The linearity was consistent (r2 = 0.9987) and recovery was more than 85.0% for both analyses. CONCLUSION: This method is applicable for routine monitoring of patients' VRZ plasma level with fast and accurate runtime to assess CYP2C19 genotype.

Determination of plasma Levetiracetam level by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS-MS) and its application in pharmacokinetics studies in neonates.

BACKGROUND: Levetiracetam (LEV) is an antiepileptic drug which has good safety and efficacy in neonatal seizure (NS), a common incident in neonates with weight <1500 g. The pharmacokinetics for LEV in neonatal populations is yet to be clearly understood. In this study, we developed and validated a method for determination of LEV in plasma by liquid chromatography tandem mass spectrometry for the purpose of pharmacokinetic study. METHODS: Plasma LEV was spiked with Lamivudine as internal standard before extraction by C18 solid-phase extraction (SPE) cartridge. Chromatography was performed using isocratic elution with mobile phase A: B (10: 90) for 2.0 min with flow rate 0.4 mL/min. The mobile phase was composed of 0.1% formic acid in 10.0 mM ammonium acetate (A) and 100% methanol (B). The injection volume was 1.0 µL and the total run time was 2.0 min. Multiple reaction monitoring (MRM) with electro spray in positive mode was used. The mass transition for LEV was 171.2/126.0 and 230.0/112.0 for IS with retention time of 0.73 and 0.72 min, respectively. RESULTS: A calibration curve range from 0.50-80.0 µg/mL was obtained with a correlation coefficient >0.99 in the quadratic model. Precision and accuracy was within the acceptable range and the intra- and inter-day %CV for three concentrations of QCs were <10%. CONCLUSION: This method was reliable, accurate and applicable for LEV pharmacokinetic study in neonates with seizure.

Development and validation of a reliable method for thiopurine methyltransferase (TPMT) enzyme activity in human whole blood by LC-MS/MS: An application for phenotypic and genotypic correlations.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of thiopurine methyltransferase (TPMT) activity in human whole blood lysate, based on conversion of 6-mercaptopurine (6-MP) by TPMT to 6-methylmercaptopurine (6-MMP) using S-adenosyl-l-methionine (SAM) as the methyl donor. This method was improved from the previous laborious method for washing of red cell lysate preparation to develop whole blood EDTA lysate. In addition, the TPMT incubation was optimized and the chromatography was performed in a short runtime of 7min on a C18-column by detection via triple quadrupole mass spectrometry. The MS/MS was optimally tuned to monitor mass to charge a ratio (m/z) for 6-MMP 167.2→151.9 and the isotope 6-MMP-d3 with m/z of 170.5→152.2 were applied as an internal standard. The calibration curve covered the range of 2.5-360ng/ml and the correlation coefficient was greater than 0.999. The accuracy of this method was determined in four concentrations of control of quality that ranged between 99.33 and 106.33%. The intra-assay coefficient of variation (CV) was less than 4.41% and the inter-assay was less than 5.43%. This method developed for measuring TPMT by LC-MS/MS is a reliable, safe, and simple with a small volume requirement (100µl of whole blood EDTA). The assay was used to study TPMT activity in 132 Thai children with a range from 29.0 to 89.1nmol 6-MMP/g Hb/h with means and median values of TPMT activity 55.9±12.47nmol 6-MMP/g Hb/h and 54.2nmol 6-MMP/g Hb/h. The genotype-phenotype association of TPMT was evaluated for common ethnic Thai single nucleotide polymorphisms (SNP) in 30 samples and demonstrated good concordance.